Traditional automated phenotypic ID tests, such as the Vitek®, Phoenix and Microscan® systems, or manual phenotypic tests such as API require that microorganisms be in an appropriate growth phase and free of interfering media and blood products in order to provide robust results. These systems use colonies grown from the positive broth for 18-24 hours on plated media. However, in an effort to obtain faster results, some laboratories have reported using these systems with microorganisms isolated from clinical samples. Faster and more broadly specific tests are urgently needed in order to provide the physician with clinically relevant results.
Identifying microorganisms cultured in liquid media is particularly difficult because of the lower concentration of microorganisms in the sample container and because the liquid media may interfere with analytical methods such as mass spectrometry.
Mass spectrometric methods have the potential to allow for identification of microorganisms very quickly, but may encounter interference from the many compounds present in liquid culture media and in clinical samples such as sputum, sterile body fluids, or combinations thereof.
Other methods for characterization and/or identification of microorganisms have been described, including:
U.S. Pat. No. 6,177,266, which discloses a method for the chemotaxonomic classification of bacteria with genus, species and strain specific biomarkers generated by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of either cellular protein extracts or whole cells.
U.S. Pat. No. 8,735,091, which discloses a method for the inactivation and extraction of acid-fast bacteria, such as Mycobacterium and Nocardia, from solid and liquid media. The '091 patent, however, does not recognize the difficulty associated with the protein extraction of acid-fast bacteria when grown in liquid media. In particular, the '091 patent does not recognize the difficulty in securing a sufficient amount of biomass of microorganisms from liquid media for identification using mass spectrometry. Further, the '091 patent does not recognize the difficulty in removing the inactivating solution, which interferes with identification using mass spectrometry. The '091 patent does not recognize the unexpected benefits of collection and retention of sufficient biomass of the acid fast bacteria for the inactivation and extraction in a tube having a specific size and/or shape. Finally, the '091 patent does not address the difficulty of separating the pellet from liquid media to avoid interference for identification using mass spectrometry.
Thus, there remains a need in the art for efficient and rapid protocols for the inactivation and/or extraction of microorganisms from liquid media for subsequent analysis, characterization and/or identification by mass spectrometry. In particular, inactivation, or cell death, is often necessary for subsequent handling of acid-fast bacteria, such as Mycobacterium and Nocardia, outside a Biosafety Level-3 (BSL-3/P3) environment.